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为了研制一种安全有效的、能预防A群链球菌引起的急性风湿热的菌苗,作者采用聚合酶链反应(PCR)技术分别扩增24、5、6、19、1、3、18和2型A群链球菌的emm基因,分别纯化8个扩增片段后,通过限制酶切位点连接扩增产物,将构建的八价基因与载体pkk223-3联接,转染大肠杆菌JM105株,利用聚丙烯酰胺凝胶电泳从大肠杆菌周质提取液中提取重组八价蛋白.用免疫印迹法检测纯化八价蛋白的性质,用ELISA法测定重组蛋白免疫家兔后诱生的抗体滴度,用体外调理试验和间接杀菌试验检测抗体的调理作用,用间接免疫荧光法检测免疫血清与人是否存在交叉反应.结果显示,八价M蛋白的分子量约为
In order to develop a safe and effective vaccine against acute rheumatic fever caused by Streptococcus group A, the authors used polymerase chain reaction (PCR) to amplify 24,5,6,19,1,3,18 and 2 group A streptococcus emm gene were purified 8 amplified fragments were amplified by restriction enzyme cleavage site, the constructed gene was linked to the vector pkk223-3, transfected into E. coli JM105 strain, Polyacrylamide gel electrophoresis was used to extract the recombinant octavalent protein from E.coli periplasmic extract.The purified octavalent protein was detected by immunoblotting and the antibody titer induced by recombinant protein was detected by ELISA, In vitro conditioning test and indirect bactericidal test were used to detect the effect of antibody and indirect immunofluorescence assay was used to detect whether there was cross-reaction between immune serum and human.The results showed that the molecular weight of the eight-valent M protein was about